Objective:To investigate the effects of siRNAs targeting CD97 immune epitopes on proliferation, infiltration, apoptosis and cell cycle of breast cancer cells. Methods:siRNA sequences targeting CD97^EGF and CD97^Stalk immune epitopes were designed according to Gene Bank NM_001025160.2 with smart siCatch^TM siRNA design software. CD97siRNAs were transfected into MDA-MB231 cells in which CD97 was highly expressed. Highest sensitive CD97^EGF and CD97^Stalk siRNA were screened by Western blotting. Inverted microscope was used to observe the growth of CD97siRNAs-transfected MDA-MB231 cells; the proliferation activity of MDA-MB231 cells was detected by MTT method; the wound healing assay and Transwell migration test were performed to examine the migration and infiltration ability of CD97^EGF and CD97^Stalk siRNA-transfected MDA-MB231 cells; the effects of CD97^EGF siRNA and CD97^Stalk siRNA on cell apoptosis and cell cycle of MDA-MB231 cells were detected by TUNEL and flow cytometry. Results:The growth and proliferation activity of CD97siRNAs-transfected MDA-MB231 cells were significantly lower than those in the control groups, and such differences were more significant in CD97^Stalk siRNA-transfected group (all P<0.05); scratch test showed that the wound healing rate was lower in CD97siRNAs-transfected groups, especially in CD97^Stalk siRNA-transfected group (all P<0.05); Transwell migration showed that the number of MDA-MB231 cells crossing through chambers were less in CD97siRNAs-transfected groups, especially in CD97^Stalk siRNA-transfected group (all P<0.05); no significant difference in cell apoptosis was observed between CD97siRNAs-transfected groups and control groups; cell cycle detection showed that CD97siRNAs-transfected groups had less cells in G₀/G₁ phase and more cells in S phase compared with the control groups, and such effect on cell cycle was more marked in CD97^Stalk siRNA-transfected group (all P<0.05). Conclusion:CD97 plays an important role in the cell growth, proliferation, migration and invasion of breast cancer MDA-MB231 cells, and compared with CD97^EGF, CD97^Stalk may have more effective inhibitory effects on cellular malignant behaviors.

Objective:To investigate the effects of microRNA(miRNA)-29b on the proliferation and migration of breast cancer cells and its molecular mechanism. Methods:The recombinant lentiviral expression vector (lenti-miRNA-29b) was constructed and transfected into 293T cells to obtain lentivirus particles that were used to infect breast cancer MCF-7 cells. Transfection efficiency of lenti-miRNA-29b in MCF-7 cells was identified by the expression of green fluorescent protein (GFP). The expression of miRNA-29b was detected by real-time PCR. The cell proliferation and migration were detected by CCK8 assay and Transwell assay, respectively. The bioinformatics softwares were used to predict and screen the downstream target genes regulated by miRNA-29b, which were verified by double luciferase reporter gene assay, RT-PCR and Western blot. The effects of screened target gene RTKN on the growth and migration of MCF-7 cells were verified by RTKN siRNA. Results:Recombinant lentiviral expression vector of miRNA-29b were successfully constructed. About 90% and 60% of the breast cancer cells showed green fluorescence in lenti-miRNA-29b and lenti-miRNA-NC groups, respectively. The expression of miRNA-29b in lenti-miRNA-29b group increased significantly compared with the lenti-miRNA-NC group and blank control group (all P<0.05); the proliferation and migration ability of MCF-7 cells significantly reduced compared with the control group (all P<0.05). The screening with bioinformatics softwares found that the 3'UTR coding region RTKN had the binding site to miRNA-29b; the dual luciferase reporter gene assay showed that the luciferase activity decreased significantly after the MCF-7 cells were co-transfected with wild type RTKN-WT-3'UTR and miRNA-29b mimics report gene vector (P<0.05). The RTKN proteins in MCF-7 cells were significantly decreased after transfection with siRNA-RTKN, and the proliferation and migration ability of MCF-7 cells were significantly reduced (all P<0.05). Conclusion:MiRNA-29b can inhibit the proliferation, invasion and metastasis of breast cancer cells by inhibiting the expression of RTKN.

Objective:To investigate the effect of CXC chemokine receptor 4 (CXCR4) on cell cycle of breast cancer and its molecular mechanisms. Methods:The expression of CXCR4 and S phase kinase associated protein 2 (Skp2) was detected by real-time fluorescence quantitative PCR (fqRT-PCR) and Western blot in breast cancer cells. The expression of signal proteins and the downstream genes of Skp2 was detected by Western blot. The effect of CXCR4, PI3K/Akt pathway inhibitor LY294002 and ERK pathway inhibitor U0126 on cell cycle of breast cancer was detected by propidium iodide staining. Results:Skp2 was significantly down-regulated in CXCR4-downregulated cells and up-regulated in CXCR4-upregulated cells. CXCR4 also regulated the expression of Skp2 and other downstream genes by signaling protein. The proportion of cells in G₀/G₁ phase increased and that in S phase declined in CXCR4-downregulated cell, and the effect was more significant when combined with the use of LY294002 or U0126. Conclusion:CXCR4 can affect cell cycle and inhibit the proliferation of breast cancer cells by regulating Skp2 gene expression through PI3K/Akt and ERK signaling pathway.

Objective:To investigate the effects of long non-coding RNA(lncRNA) RP11-770J1.3 and transmembrane protein 25 (TMEM25) on paclitaxel resistance in human breast cancer MCF-7/PR cell line. Methods:The expression of lncRNA RP11-770J1.3 and TMEM25 in human breast cancer MCF-7(paclitaxel sensitive) and MCF-7/PR(paclitaxel resistant) cells were detected by quantitative RT-PCR. The synthetic interfering fragments of lncRNA RP11-770J1.3 and TMEM25 were transfected into MCF-7/PR cells. Sulforhodamine B assay was used to detect the sensitivity of MCF-7/PR cells to paclitaxel after interference of lncRNA RP11-770J1.3 and TMEM25. The expression of multidrug-resistance genes and proteins were detected by qRT-PCR and Western blot, respectively. Results:lncRNA RP11-770J1.3 and TMEM25 were highly expressed in MCF-7/PR cells, and were significantly down-regulated after transfection of synthetic interfering fragments. Down-regulation of lncRNA RP11-770J1.3 and TMEM25 enhanced the sensitivity of MCF-7/PR cells to paclitaxel, and inhibited the expression of MRP, BCRP and MDR1/P-gp (all P<0.05). Such effects were more significant when lncRNA RP11-770J1.3 and TMEM25 were both down-regulated (all P<0.05). Conclusion:lncRNA RP11-770J1.3 and TMEM25 are highly expressed in MCF-7/PR cells, and the down-regulation of lncRNA RP11-770J1.3 and TMEM25 can enhance paclitaxel sensitivity in MCF-7/PR cells.

Objective:To evaluate the collateral flow in patients with ischemic stroke due to acute basilar artery occlusion by dynamic CT angiography and to predict the outcome after reperfusion therapy. Methods:Forty-five patients with stroke due to acute basilar artery occlusion undergoing reperfusion treatment in the Second Affiliated Hospital of Zhejiang University School of Medicine during January 2012 and August 2016 were retrospectively reviewed. Univariate and binary logistic regression model were used to identify the independent predictors of patient's outcome, and the receiver operating characteristic (ROC) curve was used to determine the optimal threshold of the posterior circulation collateral score (PC-CS) in predicting the prognosis of the patients. Results:Binary logistic regression analysis indicated that the baseline National Institutes of Health Stroke Scale (NIHSS) score (OR=0.886, 95% CI:0.802-0.979, P<0.05) and PC-CS (OR=1.962, 95%CI:1.026-3.752, P<0.05) were independent predictors of patient's outcome, and PC-CS 4.5 was the optimal threshold (AUC:0.837, sensitivity of 68.2%, specificity of 87.0%). Conclusion:Dynamic CT angiography based on CT perfusion imaging can be used to evaluate collaterals in posterior circulation, and to predict clinical outcome after reperfusion therapy in patients with acute basilar artery occlusion.

Objective:To evaluate the value of collateral score based on CT perfusion (CTP-CS) in predicting the clinical outcome of patients with anterior circulation ischemic stroke after thrombectomy. Methods:Clinical data of acute ischemic stroke patients with anterior artery occlusion undergoing endovascular treatment in the Second Affiliated Hospital, Zhejiang University School of Medicine during October 2013 and October 2016 were retrospectively reviewed. Collateral scores were assessed based on CTP and digital subtraction angiography (DSA) images, respectively. And DSA-CS or CTP-CS 3-4 was defined as good collateral vessels. Good clinical outcome was defined as a modified Rankin Scale (mRS) ≤ 2 at 3 months after stroke. The binary logistic regression model was used to analyze the correlation between the collateral score and clinical outcome, and the receiver operating characteristic (ROC) curve was used to analyze the value of DSA-CS and CTP-CS in predicting the clinical outcome. Results:Among 40 patients, 33 (82.5%) acquired recanalization and 16 (40.0%) got good outcome. Compared with poor outcome group, the collateral score (all P<0.05) and the rate of good collateral vessels were higher in good outcome group (all P<0.01). After adjust baseline National Institute of Health Stroke Scale (NIHSS) and onset to recanalization time (ORT), good collateral vessels were independent factor of good outcome (CTP-CS:OR=48.404, 95% CI:1.373-1706.585, P<0.05; DSA-CS:OR=34.651, 95% CI:1.147-1047.018, P<0.05). Collateral scores based on CTP and DSA had good consistency (κ=0.697, P<0.01), and ROC curve showed that the predictive value of CTP-CS and DSA-CS were comparable (both AUC=0.726, 95%CI:0.559-0.893, P<0.05). Conclusion:CTP-CS can predict the clinical outcome of patients with anterior circulation ischemic stroke after thrombectomy.

Objective:To investigate the relationship between maximal infarct volume to benefit from intravenous thrombolysis (IVT) and onset to needle time (ONT). Methods:The clinical and image data of acute ischemic stroke patients who received IVT in the second Affiliated Hospital, Zhejiang University School of Medicine during May 2009 to June 2016 were retrospectively reviewed. Patients were classified into within-time-window group (ONT ≤ 4.5 h) and beyond-time-window group (ONT>4.5 h). Good and poor outcome were defined as modified Rankin scale (mRS) ≤ 2 or >2 at 3 months, respectively. The maximal infarct volume was analyzed by receiver operating characteristic (ROC) curve. Results:Among 587 patients (465 cases were within-time-window, 122 cases were beyond-time-window), baseline core volume was 15(2-46)mL,and 324 (55.2%) patients achieved good outcome. Compared with the good-outcome group, the baseline core volume was larger in the poor-outcome group (32 mL vs 5 mL,Z=-9.766,P<0.01). After adjusting age, ONT, baseline National Institutes of Health Stroke Scale (NIHSS) and atrial fibrillation, baseline infarct core volume independently predicted poor outcome (OR=1.014, 95% CI:1.008-1.020, P<0.01). The ROC curve analysis showed that the maximal infarct core volume for achieving good outcome in the within-time-window group and beyond-time-window group were 152 mL and 71mL, respectively. The maximal infarct volume to benefit from IVT diminished with the increasing delayed ONT of every 30 min (ρ=-0.691, P<0.05). Conclusion:The maximal infarct volume to benefit from thrombolysis is larger in patients treated within time window than those beyond the time window, and that volume diminishes with ONT delay.

Objective:To investigate the association of serum folate level with the severity of white matter hyperintensity (WMH) and presence of cerebral microbleeds (CMB). Methods:Clinical data of WMH patients from the second affiliated Hospital, Zhejiang University school of Medicine during July 2011 and February 2016 were retrospectively reviewed. According to Fazekas score based on T2-Flair images, patients were classified into mild WMH (0-3) and severe WMH (4-6). The presence of CMB was assessed on susceptibility weighted imaging (SWI). Binary logistic analysis was conducted to identify the independent predictors for severe WMH and the presence of CMB. Results:Two hundred and twenty eight patients with WMH were included, among whom 149(65.35%)had severe WMH. In patients with high folate (≥ 15.68 nmol/L), low folate (6.8-15.67 nmol/L) and folate deficiency (<6.8 nmol/L), the proportions of severe WMH were 52.88%, 73.33% and 89.47%, respectively. Binary logistic regression analysis revealed that compared with high folate group, severe WMH was more common in groups with low folate (OR=2.109, 95% CI:1.112-4.001,P<0.05) and folate deficiency (OR=6.383, 95% CI:1.168-34.866, P<0.05). Eighty-eight(48.09%) of 183 patients receiving SWI scan presented with CMB. Although the subjects with CMB had lower serum folate level than those without CMB(13.42 vs 16.51 nmol/L, P<0.01), binary logistic regression analysis did not reveal the independent association between serum folate level and the presence of CMB after adjusting for hyperhomocysteinemia (P>0.05). Conclusion:Lower serum folate level is independently associated with severe WMH, but not with the CMB concurrence.

Objective:To investigate the effect of tirofiban on hemorrhagic transformation and neurological outcome in patients with acute cerebral infarct treated with endovascular therapy. Methods:One hundred and fifteen patients with acute cerebral infarct who received endovascular stent mechanical thrombectomy in the Second Affiliated Hospital, Zhejiang University School of Medicine during October 2013 and April 2017 were included in the study. Among 115 patients, 30 received tirofiban treatment. Hemorrhagic transformation and neurological outcomes were assessed using the ECASS Ⅱ criteria and modified Rankin scale (mRS), respectively. Unfavorable outcome was defined as mRS>2. Binary logistic regression model was used to analyze the independent predictors of hemorrhagic transformation and neurological outcome. Results:Binary logistic regression analysis showed that tirofiban treatment did not increase the risk of hemorrhagic transformation (OR=0.437, 95% CI:0.168-1.132, P>0.05); baseline NIHSS (OR=1.136, 95% CI:1.014-1.273, P<0.05), recanalization (OR=0.060, 95% CI:0.010-0.365, P<0.01), hypertension (OR=4.233, 95% CI:1.320-13.570, P<0.05) and onset to treatment time(OR=1.006, 95% CI:1.001-1.011, P<0.05) were independently associated with unfavorable outcome, while such association was not observed in tirofiban treatment (OR=1.923, 95% CI:0.536-6.568, P>0.05). Conclusion:Tirofiban appears to be safe for patients with acute cerebral infarct receiving endovascular therapy.

Objective:To investigate the effect of mammalian target of rapamycin(mTOR) inhibitor-rapamycin on cognitive function after chronic cerebral ischemia in mice and its molecular mechanism. Methods:The chronic cerebral ischemia model was induced by ligation of right common carotid artery (rUCCAO) in 6-week-old ICR mice. The expressions of mTOR, S6K, S6 and corresponding phosphorylated proteins were detected by Western blotting at different time interval (1 h, 3 h, 6 h, 24 h, 3 d, 7 d, 2 w, 4 w, 6 w) after rUCCAO to determine the changes of mTOR signaling pathway. Rapamycin was administrated i.p. at the dose of 3.0 mg/kg 24 h after rUCCAO. Fluoro Jade B staining was used to detect the apoptotic cells. The expressions of Beclin and LC3-Ⅱ were detected by Western blotting to determine the status of autophagy. Morris water maze test and Y maze test were performed to evaluate cognitive functions. Results:The mTOR signaling pathway was abnormally activated from 6 h to 6 w after rUCCAO in mouse cortex. The activation of mTOR signaling pathway induced by rUCCAO was reversed by administration of rapamycin, and the apoptotic cell number was significantly decreased (146.1±16.3 vs 84.5±9.6, P<0.05). Meanwhile, the elevation of Beclin and LC3-Ⅱ protein induced by rUCCAO was reversed by rapamycin administration. Furthermore, compared with vehicle-treated mice, the latent period[(11.1±2.3) s vs (8.1±1.8) s, P<0.05] and swimming distance[(672.8±128.5) cm vs (558.2±124.9) cm, P<0.05] were significantly decreased and the number of crossing the platform quadrant in Morris water maze increased(2.8±0.9 vs 5.2±0.8, P<0.05) in rapamycin-treated mice. Correct response rate in the Y maze was also increased significantly in rapamycin-treated mice[(38.5±9.2)% vs (64.9±7.9)%, P<0.05]. Conclusion:Inhibiting mTOR pathway by rapamycin reverses the rUCCAO-induced cognitive impairment partly through the suppression of apoptosis and autophagy.

Objective:To investigate the drug resistance, β-lactamase-encoding genes and protoporphyrin ferrochelatase-encoding genes of Ralstonia mannitolilytica, and to explore its structure and pathogenic function. Methods:The strain was isolated by plate streaking method and identified by automatic bacteria detection system and 16S RNA gene PCR. Microdilution method was applied for drug susceptibility test. β-lactamases, extended spectrum β-lactamases (ESBL) and carbapenemases were detected using nitrocefin-disk, Kirby-Bauer disk, and Hodge test, respectively. Five β-lactamase-encoding genes and protoporphyrin ferrochelatase-encoding gene of the isolate were amplified by PCR for sequencing. Bioinformatic softwares were used to analyze the structure and function of the product of protoporphyrin ferrochelatase-encoding gene. Results:A strain belonging to Ralstonia mannitolilytica was isolated. This isolate was sensitive to cefepime, ciprofloxacin, ofloxacin and tigecycline, but resistant to five penicillins, four cephalosporins and two carbapenems antibiotics. The isolate produced β-lactamases but did not produce ESBL and carbapenemases. The isolate had five distinct β-lactamase-encoding genes and protoporphyrin ferrochelatase-encoding gene. The product of protoporphyrin ferrochelatase-encoding gene contained two functional domains of protoporphyrin ferrochelatase belonging to type Ⅱ chelatase superfamily that presented the most closely genetic relationship with the protoporphyrin ferrochelatase of Neisseria meningidis. Conclusion:The isolate of Ralstonia mannitolilytica has a higher resistance to β-lactam antibiotics and its β-lactamase-encoding genes are different with the common bacterial β-lactamase-encoding genes. Protoporphyrin ferrochelatase may act as an important virulence factor of Ralstonia mannitolilytica.

Objective:To evaluate the efficacy and safety of humanized anti-IL-6 receptor monoclonal antibody (tocilizumab) in treatment of systemic juvenile idiopathic arthritis (sJIA). Methods:Thirteen sJIA patients admitted between December 2015 and November 2016 and received tocilizumab treatment were enrolled in the study. The complete blood count (CBC), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), interleukin-6 (IL-6) and ferritin levels were measured; American College of Rheumatology Pediatric(ACR Pedi)30/50/70/90 scores were assessed; and the use of glucocorticosteroid and adverse events were documented. Results:Compared with the baseline levels, the CRP and ESR at d3 were decreased (all P<0.05); hemoglobin was increased and platelet was decreased at week 2 (all P<0.05), ferritin decreased at week 4, white blood cell (WBC) decreased at week 8 after treatment with tocilizumab (all P<0.05). The level of IL-6 was rising at d3 and week 2 and descending at week 4, but no significant difference was observed compared with the baseline level (all P>0.05). All 13 patients achieved ACR Pedi 30 remission at week 4, 61.5% achieved ACR Pedi 90 remission and glucocorticosteroids were withdrawn at week 20. Twenty two adverse events occurred, and infection accounted for 54.5% (12/22); no severe adverse reactions were observed during 20-week follow-up. Conclusion:Tocilizumab is safe and effective in treatment of sJIA, with decreasing inflammation, improving disease activity and reducing glucocorticosteroid use.

Objective:To develop an all-in-one CRISPR/Cas9 vector system that can efficiently knockdown miR-101a expression in mice.Methods:Three sgRNAs targeting mouse miR-101a gene and a small guide (sgRNA) targeting green fluorescent protein gene were designed and constructed into an all-in-one vector system (pENTRY-U6-sgRNA-WT Cas9). Moreover, sgRNA1 and sgRNA3 were selected and constructed into a double-nicking Cas9 vector (pENTRY-U6-sgRNA-U6-sgRNA-Cas9 D10A). The constructed plasmids were transfected into mouse liver AML12 cells for validation by T7 EndoⅠ(T7EⅠ) 72 h after transfection. The pAD vectors were cloned via the Gateway system, and the recombinant adenovirus vectors were packaged in 293A cells. The virus particles were used to infect AML12 cells and the expression levels of mature miR-101a were analyzed to monitor the knockout efficiency after 72 h. Results:The constructed pENTRY all-in-one vectors were validated by gene sequencing and T7EⅠ assay, which showed CRISPR/Cas9-mediated mismatches at target sites of miR-101a gene. The adenovirus vectors were constructed successfully. The CRISPR/Cas9 containing adenovirus was introduced to AML12 cells and the quantitative real-time PCR assays indicated that the expression level of mature miR-101a was significantly decreased compared with that of the control (all P<0.01). Conclusions:We have successfully constructed two "all-in-one" CRISPR/Cas9 vector systems targeting miR-101a gene in mouse liver AML12 cells with high efficiency. It provides experimental basis for research of microRNA, and a reference method for knockout of other miRNAs.

Objective:To investigate the effect of low-salt diet on gene expression of canine cardiac tissue. Methods:Microarray data (GSE17149) of heart tissue from dogs fed with low-salt diet was obtained from the Gene Expression Omnibus (GEO). The data were analyzed by Qlucore Omics Explorer 3.1, STRING 10.0, Genclip 2.0 and GCBI. The protein-protein interactions (PPI) of the differentially expressed genes in low-salt and control groups were conducted to screen the key node genes between the two groups. Results:Compared with the control group, the gene expression profile of the dog heart tissue was changed in the low-salt diet group, and 1343 (3.12%) differentially expressed genes were found in 43 035 genes. The differentially expressed gene protein interaction analysis revealed a most obvious protein (NFKBIA) as the core, which might be related to inhibiting the differentiation of macrophage-derived foam cell and promoting cholesterol discharge and decomposition. Conclusion:Gene expression of heart tissue in dogs fed with low-salt diet is significantly changed and low-salt diet may reduce the risk of cardiovascular disease by up-regulating the expression of NFKBIA.

Objective:To investigate the effects of the volume of 4% neutral phosphate buffered formalin fixative solution on the detection of human epidermal growth factor receptor 2 (HER2) gene by fluorescence in situ hybridization (FISH) in primary invasive breast cancer. Methods:Tissue samples were collected from 109 patients with primary invasive breast cancer admitted in Zhongshan Boai Hospital from June 2014 to October 2016. The ratios of 4% phosphate buffered formalin fixative solution to sample volume samples were 3:1, 6:1, 9:1, 10:1, 15:1, 20:1 or 25:1 (groups A, B, C, D, E, F and G), respectively. Paraffin sections were made after 15 h of fixation. The amplification of HER2 gene was detected by FISH. The gene amplification results of HER2 were observed and compared in different groups. Results:Fluorescence microscope showed that the tissue contour in groups A, B and C was vague, cell debris appeared, and the probe was positioned poorly; while the tissue contour was clear and complete in groups D, E, F and G and the probe was positioned accurately. The positive rate of HER2 was gradually increased from group A to D(χ²=8.601, P<0.01), and that remained stable at 24.77% in groups D to G. The positive rate of gene amplification in groups D, E, F and G was significantly higher than that in groups A, B and C (all P<0.05). Conclusion:When using FISH to detect HER2 gene in samples of primary breast invasive carcinoma, the volume of fixative solution should be at least 10 times of the sample volume to obtain accurate and stable results.

Two cases of hypoglossal canal dural arteriovenous fistulas (HCDAVF) were reported. The clinical manifestation, radiological features, treatment and prognosis were reviewed. Both cases presented chemosis and pulsatile tinnitus. 3D-time-of-flight (TOF) magnetic resonance angiography (MRA) demonstrated abnormal high signal in hypoglossal canal. Cerebral digital subtraction angiography (DSA) showed that these HCDAVFs were supplied by multiple intracranial and extracranial arteries, and fistulas were located in hypoglossal canal. Fistulas were blocked by coils and Onyx-18 through transvenous approach, and the angiography after the embolism showed complete occlusion of fistula. No adverse events after treatment and no recurrence during the follow up were observed.

Autoinflammatory diseases (AID) in childhood is one of refractory diseases, whose pathogenesis is not completely clear. In recent years, a large number of studies have shown that NLRP3 inflammasome plays an important role in the development of AIDs in children. Inflammasome is a cytosolic multiprotein complex that can activate cysteinyl aspartate-specific protease-1 (caspase-1), to further promote the maturation and secretion of proinflammatory cytokines IL-1β and IL-18 as well as pyroptosis and regulate innate immune response. IL-1 receptor antagonist (Anakinra) and IL-1β monoclonal antibody (Canakinumab) have good therapeutic effects in children with AIDs. This article reviews the research progress of NLRP3 inflammasome in the pathogenesis of autoinflammatory diseases.